Fluorescence microscopy is an important tool in the biomedical sciences allowing for the imaging of biological cells and tissues. One limit of fluorescence microscopy is that the optics of a microscope cannot create illuminated spots smaller than the diffraction limit, thus limiting the usefulness of such techniques to image biological samples at high resolution, generally below about 200 nm for visible light. The term ‘Superresolution microscopy’, also referred to as sub-diffraction limit microscopy, refers to techniques suitable for imaging objects smaller than about 200 nm.
One of the most well known Super Resolution Microscopy techniques is the stimulated emission depletion technique (STED) developed by Dr. Stefan Hell. The STED technique is a nonlinear optics technique using two laser pulses in which a first diffraction limited pulse excites fluorophores in a spot and a second “donut profile” overlapping pulse stimulates emission while simultaneously driving the fluorophores back to the ground state, effectively depleting the edges of the diffraction limited spot while allowing the center of the original spot to fluoresce. The result is a narrowed point spread function that has been shown to provide spot sizes of 20 nm or less, allowing for resolution of structures well below the diffraction limit. Typical fluorescent dyes used in STED include ATTO 647N and ATTO 655. One disadvantage of the STED technique is that large powers are generally required to narrow the point spread function, potentially damaging biological samples and limiting its usefulness.
Additional Super Resolution Microscopy techniques include stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM). Both STORM and PALM use photoactivatable probes that are activated by light. Because photoactivation is stochastic, only a few, well-separated (spaced beyond Rayleigh criterion/Abbe limit) molecules are photoactivated with each pulse of light. After registration (through repeated luminescence excitation/emission cycles) and photobleaching of the activated spots, another flash of photoactivating light generates another different subpopulation of photoactivatable molecules. The process is repeated many times, fitting the point spread functions to obtain precise center loci and building up an image molecule-by-molecule, a “pointillist” approach. Because the molecules were localized at different times, the apparent resolution of the final image can be much higher than that limited by diffraction. A drawback of both STORM and PALM is that it may take several minutes or even hours to collect the data needed to produce images.
Super Resolution Microscopy techniques that allow for improved ultrafine imaging, particularly of living biological samples, would be desirable.